Method of inhibiting cell growth with the P2U receptor

ABSTRACT

Isolated DNAs encoding the human P2U receptor are disclosed, along with vectors and host cells containing the same and methods of using the same. Host cells which are essentially free of endogenous P2U receptor expression, and which express a heterologous P2U receptor such as a murine P2U receptor, are also disclosed, along with methods of using the same.

This invention was made with Government support under Grant Nos. DHHS 5R01 DE07389-08 and 5-P01-HL34322 awarded by the NIH. The Government hascertain rights in the invention.

This application is a divisional of prior application Ser. No.08/138,137, filed Oct. 15, 1993, now abandoned, the disclosure of whichis incorporated herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to DNA encoding P_(2U) receptors, andparticularly relates to DNA encoding the human P_(2U) receptor, and nullcells which express heterologous P_(2U) receptors.

BACKGROUND OF THE INVENTION

Cystic fibrosis (CF) exhibits reduced Cl⁻ secretion by airway epithelia.Consequently, one of the most debilitating effects of CF is thedevelopment of a dehydrated, viscous mucus which obstructs the airwaysand compromises lung function. Extracellular nucleotide triphosphates,such as ATP or UTP, are able to regulate Cl⁻ secretion in human airwayepithelia and, in combination with an inhibitor of Na⁺ transport, mayprovide an alternative, non-CFTR-dependent mechanism to induce fluidsecretion in CF airway epithelia. Extracellular nucleotides alsostimulate mucus secretion by goblet cells in vitro and excessiveactivation of this pathway in vivo may be partly responsible for thehypersecretion observed in chronic bronchitis. In both cases, theresponses are mediated by 5'-nucleotide (P_(2U)) receptors on the cellsurface.

Recently, a cDNA encoding a murine ATP/UTP receptor was cloned fromneuroblastoma cells by functional expression in Xenopus oocytes (K.Lustig et al., Proc. Natl. Acad. Sci. USA 90, 5113 (1993)). Thereceptor, a member of the G protein-coupled receptor superfamily, isactivated by UTP and ATP, initiates elevation of cytoplasmic calcium,and has been identified with the subtype of P₂ -receptor provisionallydesignated P2U. Its pharmacological and signaling properties are verysimilar to those described for a 5'-nucleotide (P_(2U)) receptor presentin the human airway epithelial cell line, CF/T43, which was derived froma donor with CF (A. Brown et al., Mol. Pharmacol. 40, 648 (1991)).

Isolation and molecular characterization of the receptor forextracellular nucleotides present in human airway epithelia will permitstudies of the expression of this receptor in normal and diseasedtissues and facilitate identification of new drugs for therapy.

SUMMARY OF THE INVENTION

In view of the foregoing, a first aspect of the present invention isisolated DNA encoding an P_(2U) receptor selected from the groupconsisting of: (a) isolated DNA (e.g., the DNA of SEQ ID NO:1) whichencodes the human P_(2U) receptor having the amino acid sequence givenherein as SEQ ID NO:2; (b) isolated DNA which hybridizes to isolated DNAof (a) above and which encodes a human P_(2U) receptor; and (c) isolatedDNA differing from the isolated DNAs of (a) and (b) above in nucleotidesequence due to the degeneracy of the genetic code, and which encodes ahuman P_(2U) receptor.

A second aspect of the present invention is a recombinant DNA sequencecomprising vector DNA and a DNA as given above which encodes an P_(2U)receptor.

A third aspect of the present invention is a host cell containing arecombinant DNA sequence as given above and capable of expressing theencoded P_(2U) receptor.

A fourth aspect of the present invention is isolated DNA as given above,and oligonucleotides as given above, configured in antisense for theproduction of antisense RNA which inhibits the expression of P_(2U)receptor. (hereinafter referred to as "antisense DNAs"). Such antisenseDNAs may be provided in a vector as given herein for transcription in asuitable cell where they then inhibit the production of the P_(2U)receptor. In the alternative, antisense oligonucleotides which bind toRNA in cells and inhibit the expression of P_(2U) receptor therein maybe delivered directly to cells.

A fifth aspect of the present invention is a transformed null cell whichis essentially free of endogenous P_(2U) receptor expression, which nullcell contains and expresses heterogenous DNA encoding an P_(2U)receptor, said heterogenous DNA selected from the group consisting of:(a) isolated DNA consisting essentially of DNA which encodes the humanP_(2U) receptor having the amino acid sequence given herein as SEQ IDNO:2; (b) isolated DNA which hybridizes to isolated DNA of (a) above andwhich encodes a P_(2U) receptor; and (c) isolated DNA differing from theisolated DNAs of (a) and (b) above in nucleotide sequence due to thedegeneracy of the genetic code, and which encodes a P_(2U) receptor.

A sixth aspect of the present invention is a method of detectingcompounds which bind to the P_(2U) receptor. The method comprises: (a)providing a cell which contains and expresses heterogenous DNA encodinga heterogenous P_(2U) receptor, which cell secretes endogenous ATP inresponse to stimulation of said P_(2U) receptor, the heterogenous DNAbeing as described above; (b) removing essentially all endogenous ATPsecreted by said cell from said cell; (c) contacting a compound to saidcell; and then (d) detecting the binding of the compound to the P_(2U)receptor (e.g., by detecting an elevation in intracellular calciumtherein).

A seventh aspect of the present invention is a method of enhancing cellgrowth, comprising transforming a cell with a vector capable ofexpressing a P_(2U) receptor in the cell in an amount sufficient toenhance cell growth by autostimulation of the P_(2U) receptor, where thecell secretes ATP which stimulates the P_(2U) receptor, and where cellgrowth is enhanced by stimulation of the P_(2U) receptor. The method isuseful, among other things, for stimulating the growth of cultures ofmammalian cells in vitro where the cells are difficult to grow orotherwise grow slowly in culture.

An eighth aspect of the present invention is a method of inhibiting cellgrowth, comprising transforming a cell with a vector capable ofexpressing a P_(2U) receptor in the cell in an amount sufficient toinhibit cell growth by autostimulation of the P_(2U) receptor, where thecell secretes ATP, and where cell growth is inhibited by stimulation ofthe P_(2U) receptor. The method is useful, among other things, forinhibiting the growth of cells in vitro or in vivo such as inproliferative or hyperplastic diseases, both malignant and nonmalignant,of human and animal subjects.

A ninth aspect of the present invention is a method of treating diseasesof epithelial mucosal surfaces (particularly diseases of airwayepithelial mucosal surfaces such as cystic fibrosis, asthma, and chronicbronchitis) in a human or animal subject in need of such treatment byenhancing or inhibiting mucus production in that subject. The method iscarried out by administering to the subject a vector capable of enteringlung epithelial cells. The vector may be either:

(a) a vector which carries a DNA encoding a P_(2U) receptor as describedherein, which DNA is operably associated with a promoter which expressesthat gene in the epithelial cells; or

(b) a vector containing an antisense nucleic acid which encodes anantisense RNA, which antisense RNA is capable of binding to pre-mRNAencoding a P_(2U) receptor and inhibiting expression thereof in theepithelial cells.

Also disclosed herein is a vector as described above, pharmaceuticalformulations containing such a vector, and the use of such vectors forthe preparation of a medicament the treatment of epithelial mucosaldiseases in a subject in need of such treatment.

The foregoing and other aspects of the present invention are explainedin detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows ³ H! inositol phosphate production in cpm×10⁻³ in wildtype, LSN, and LHP2USN cells, with and without the present of apyrase incells which had not been exposed to exogenous nucleotides.

FIG. 1B shows ³ H! inositol phosphate production in cpm×10⁻³ in wildtype, LSN, and LHP2USN cells, in cells which had been exposed toexogenous carbachol, UTP, and ATP, or cells which had not been exposedto exogenous compounds (control) .

FIG. 2 shows the effect of nucleotides and nucleotide analogs on Ca²⁺!_(i) in K 562 cells stably expressing a P_(2U) receptor. Ca²⁺ !_(i) wasmeasured after treatment with the indicated concentration of UTP (filledcircles) , ATP (open circles) , ATPγS (filled triangles), BzATP (opentriangles), ATPαS (filled squares), or 2-MeSATP (open squares).

DETAILED DESCRIPTION OF THE INVENTION

Amino acid sequences disclosed herein are presented in the amino tocarboxy direction, from left to right. The amino and carboxy groups arenot presented in the sequence. Nucleotide sequences are presented hereinby single strand only, in the 5' to 3' direction, from left to right.Nucleotides and amino acids are represented herein in the mannerrecommended by the IUPAC-IUB Biochemical Nomenclature Commission, or(for amino acids) by three letter code, in accordance with 37 CFR §1.822and established usage. See, e.g., PatentIn User Manual, 99-102 (November1990)(U.S. Patent and Trademark Office, Office of the AssistantCommissioner for Patents, Washington, D.C. 20231); U.S. Pat. No.4,871,670 to Hudson et al. at Col. 3 lines 20-43 (applicantsspecifically intend that the disclosure of this and all other patentreferences cited herein be incorporated herein by reference).

Subjects treated by methods disclosed herein include both human subjectsand animal subjects (e.g., dog, cat, horse) for veterinary purposes.

A. DNAs ENCODING P_(2U) RECEPTORS AND OLIGONUCLEOTIDES THEREOF

DNAs which encode P_(2U) receptors, whether they are cDNAs or genomicDNAs, encode a G-protein coupled receptor protein which, on expressionin a suitable host cell, (a) selectively and stereospecifically bindsATP and UTP and (b) initiates the elevation of cytoplasmic calcium onbinding ATP or UTP. This definition is intended to encompass naturalallelic variations in the DNAs.

DNAs encoding P_(2U) receptors which hybridize to the DNA encoding thehuman P_(2U) receptor disclosed herein, may be of any species of origin,including murine (mouse, rat), rabbit, cat, porcine, and human, butpreferably code for an P_(2U) receptor of mammalian origin, and mostpreferably code for human P_(2U) receptors.

Hybridization conditions which will permit other DNA sequences whichcode on expression for an P_(2U) receptor to hybridize to a DNA sequenceas given herein are, in general, high stringency conditions. Forexample, hybridization of such sequences may be carried out underconditions represented by a wash stringency of 0.3M NaCl 0.03M sodiumcitrate, 0.1% SDS at 60° C. or even 70° C. to DNA disclosed herein in astandard in situ hybridization assay. (See J. Sambrook et al., MolecularCloning, A Laboratory Manual (2d Ed. 1989)(Cold Spring HarborLaboratory)). The same hybridization conditions are used to determinehybridization of oligonucleotides. In general, DNA sequences which codefor P_(2U) receptors and hybridize to the DNA sequence encoding thehuman P_(2U) receptor disclosed herein will be at least 70%, 75%, 80%,85%, 90%, or even 95% homologous or more with the sequence of the DNAencoding the human P_(2U) receptor disclosed herein.

In general, DNA sequences which encode human P_(2U) receptors whichhybridize to the DNA encoding the human P_(2U) receptor disclosed hereinwill be 93%, 94%, 95%, 96%, or even 97% homologous or more to the DNAsequence encoding the human P_(2U) receptor disclosed herein.

Further, DNA sequences which code for the same P_(2U) receptor as codedfor by the foregoing sequences, but which differ in codon sequence fromthese due to the degeneracy of the genetic code, are also an aspect ofthis invention. The degeneracy of the genetic code, which allowsdifferent nucleic acid sequences to code for the same protein orpeptide, is well known in the literature. See e.g., U.S. Pat. No.4,757,006 to Toole et al. at Col. 2, Table 1.

B. GENETIC ENGINEERING TECHNIQUES

The production of cloned genes, recombinant DNA, vectors, transformedhost cells, proteins and protein fragments by genetic engineering iswell known. See, e.g., U.S. Pat. No. 4,761,371 to Bell et al. at Col. 6line 3 to Col. 9 line 65; U.S. Pat. No. 4,877,729 to Clark et al. atCol. 4 line 38 to Col. 7 line 6; U.S. Pat. No. 4,912,038 to Schilling atCol. 3 line 26 to Col. 14 line 12; and U.S. Pat. No. 4,879,224 toWallner at Col. 6 line 8 to Col. 8 line 59.

A vector is a replicable DNA construct. Vectors are used herein eitherto amplify DNA encoding P_(2U) receptors as given herein and/or toexpress DNA which encodes P_(2U) receptors as given herein. Anexpression vector is a replicable DNA construct in which a DNA sequenceencoding a P_(2U) receptor is operably linked to suitable controlsequences capable of effecting the expression of the receptor in asuitable host. The need for such control sequences will vary dependingupon the host selected and the transformation method chosen. Generally,control sequences include a transcriptional promoter, an optionaloperator sequence to control transcription, a sequence encoding suitablemRNA ribosomal binding sites, and sequences which control thetermination of transcription and translation.

Amplification vectors do not require expression control domains. Allthat is needed is the ability to replicate in a host, usually conferredby an origin of replication, and a selection gens to facilitaterecognition of transformants.

Vectors comprise plasmids (e.g., the neomycin-resistance plasmid pP2R1for stable transfection; the plasmids pCCM6×1 or pP2R for transientexpression), viruses (e.g., adenovirus, cytomegalovirus), phage, andintegratable DNA fragments (i.e., fragments integratable into the hostgenome by recombination). The vector replicates and functionsindependently of the host genome, or may, in some instances, integrateinto the genome itself. Expression vectors should contain a promoter andRNA binding sites which are operably linked to the gene to be expressedand are operable in the host organism.

DNA regions are operably linked or operably associated when they arefunctionally related to each other. For example, a promoter is operablylinked to a coding sequence if it controls the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to permit translation.

Transformed host cells are cells which have been transformed ortransfected with vectors containing a DNA sequence as disclosed hereinconstructed using recombinant DNA techniques. Transformed host cellsordinarily express the receptor, but host cells transformed for purposesof cloning or amplifying the receptor DNA do not need to express thereceptor.

Suitable host cells include prokaryote, yeast or higher eukaryotic cellssuch as mammalian cells and insect cells. Cells derived frommulticellular organisms are a particularly suitable host for recombinantP_(2U) receptor synthesis, and mammalian cells are particularlypreferred. Propagation of such cells in cell culture has become aroutine procedure (Tissue Culture, Academic Press, Kruse and Patterson,editors (1973)). Examples of useful host cell lines are VERO and HeLacells, Chinese hamster ovary (CHO) cell lines, and WI138, BHK, COS-7,CV, and MDCK cell lines. Expression vectors for such cells ordinarilyinclude (if necessary) an origin of replication, a promoter locatedupstream from the DNA encoding the P_(2U) receptor to be expressed andoperatively associated therewith, along with a ribosome binding site, anRNA splice site (if intron-containing genomic DNA is used), apolyadenylation site, and a transcriptional termination sequence.

The transcriptional and translational control sequences in expressionvectors to be used in transforming vertebrate cells are often providedby viral sources. For example, commonly used promoters are derived frompolyoma, Adenovirus 2, and Simian Virus 40 (SV40). See, e.g., U.S. Pat.No. 4,599,308.

An origin of replication may be provided either by construction of thevector to include an exogenous origin, such as may be derived from SV40or other viral source (e.g. Polyoma, Adenovirus, VSV, or BPV), or may beprovided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter is oftensufficient. Rather than using vectors which contain viral origins ofreplication, one can transform mammalian cells by the method ofcotransformation with a selectable marker and the receptor DNA. Examplesof suitable selectable markers are dihydrofolate reductase (DHFR) orthymidine kinase. This method is further described in U.S. Pat. No.4,399,216.

Other methods suitable for adaptation to the synthesis of the P_(2U)receptor in recombinant vertebrate cell culture include those describedin M-J. Gething et al., Nature 293, 620 (1981); N. Mantei et al., Nature281, 40; A. Levinson et al., EPO Application Nos. 117,060A and 117,058A.

Host cells such as insect cells (e.g., cultured Spodoptera frugiperdacells) and expression vectors such as the baculovirus expression vector(e.g., vectors derived from Autographa californica MNPV, Trichoplusia niMNPV, Rachiplusia ou MNPV, or Galleria ou MNPV) may be employed incarrying out the present invention, as described in U.S. Pat. Nos.4,745,051 and 4,879,236 to Smith et al. In general, a baculovirusexpression vector comprises a baculovirus genome containing the gene tobe expressed inserted into the polyhedrin gene at a position rangingfrom the polyhedrin transcriptional start signal to the ATG start siteand under the transcriptional control of a baculovirus polyhedrinpromoter.

Prokaryote host cells include gram negative or gram positive organisms,for example Escherichia coli (E. coli) or Bacilli. Higher eukaryoticcells include established cell lines of mammalian origin as describedbelow. Exemplary host cells are E. coli W3110 (ATCC 27,325), E. coli B,E. coli X 1776 (ATCC 31,537), E. coli 294 (ATCC 31,446). A broad varietyof suitable prokaryotic and microbial vectors are available. E. coli istypically transformed using pBR322. Promoters most commonly used inrecombinant microbial expression vectors include the betalactamase(penicillinase) and lactose promoter systems (Chang et al., Nature 275,615 (1978); and Goeddel et al., Nature 281, 544 (1979)), a tryptophan(trp) promoter system (Goeddel et al., Nucleic Acids Res. 8, 4057 (1980)and EPO App. Publ. No. 36,776) and the tac promoter (H. De Boer et al.,Proc. Natl. Acad. Sci. USA 80, 21 (1983)). The promoter andShine-Dalgarno sequence (for prokaryotic host expression) are operablylinked to the DNA encoding the P_(2U) receptor, i.e., they arepositioned so as to promote transcription of P_(2U) receptor messengerRNA from the DNA.

Eukaryotic microbes such as yeast cultures may also be transformed withvectors carrying the isolated DNA's disclosed herein. see, e.g. U.S.Pat. No. 4,745,057. Saccharomyces cerevisiae is the most commonly usedamong lower eukaryotic host microorganisms, although a number of otherstrains are commonly available. Yeast vectors may contain an origin ofreplication from the 2 micron yeast plasmid or an autonomouslyreplicating sequence (ARS), a promoter, DNA encoding the receptor asgiven herein, sequences for polyadenylation and transcriptiontermination, and a selection gene. An exemplary plasmid is YRp7,(Stinchcomb et al., Nature 282, 39 (1979); Kingsman et al., Gene 7, 141(1979); Tschemper et al., Gene 10, 157 (1980)). Suitable promotingsequences in yeast vectors include the promoters for metallothionein,3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255, 2073(1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Req. 7,149 (1968); and Holland et al., Biochemistry 17, 4900 (1978)). Suitablevectors and promoters for use in yeast expression are further describedin R. Hitzeman et al., EPO Publn. No. 73,657.

C. USES OF DNAS ENCODING P_(2U) RECEPTORS.

P_(2U) receptors made from cloned genes in accordance with the presentinvention may be used for screening compounds for P_(2U) receptoractivity, or for determining the amount of a P_(2U) receptor agonist orantagonist such as ATP or UTP in a solution (e.g., blood plasma orserum), either for the purpose of quantitatively measuring the presenceof the agonist or antagonist in the solution (e.g., in a standardlaboratory assay), or screening for the presence or absence of thatcompound in the solution. For example, host cells may be transformedwith a vector of the present invention, P_(2U) receptors expressed inthat host, the cells lysed, and the membranes from those cells used toscreen compounds for P_(2U) receptor binding activity. Competitivebinding assays in which such procedures may be carried out are wellknown.

By selection of "null" host cells which do not ordinarily express P_(2U)receptors (e.g., cells which are essentially free of endogenous P_(2U)receptor expression, and more preferably cells which are essentiallyfree of endogenous P_(2X), P_(2Y) and P_(2U) receptor expression, whichcells may or may not be essentially free of endogenous P_(2T) receptorexpression), preparations free of other P₂ receptors which mightinterfere with the assay can be obtained. Further, P_(2U) receptoragonists and antagonists, particularly UTP and analogs thereof, can beidentified by transforming host cells with vectors of the presentinvention, which host cells also express the several G-protein subunitsand effector proteins necessary to cause the cell to initiate elevationof cytoplasmic calcium. P_(2U) receptor agonists will initiate elevationof cytoplasmic calcium when they bind to and activate receptorsexpressed in such cells. Such cells must be capable of operativelyassociating the P_(2U) receptor with the appropriate effector proteins:e.g., G protein must also be present in the cell membranes in theappropriate configuration. Such cells are typically mammalian cells,including human cells, examples being human leukemia cells (e.g., K562human leukemia cells), and human astrocytoma cells (e.g., 1321N₁ humanastrocytoma cells).

As noted above, one particular method of detecting compounds which bindto the P_(2U) receptor comprises (a) providing a cell which contains andexpresses heterogenous DNA encoding a heterogenous P_(2U) receptor,which cell secretes endogenous ATP which stimulates the P_(2U) receptor,the heterogenous DNA being as described above; (b) removing essentiallyall endogenous ATP secreted by the cell from the cell (e.g., byinactivating the endogenous ATP or by separating the endogenous ATP fromthe cell); (c) contacting a compound to the cell; and then (d) detectingthe binding of the compound to the P_(2U) receptor. The method isparticularly useful for detecting UTP and analogs thereof. Thecontacting step is typically carried out in an aqueous solution, thoughthe cells can also be immobilized on a solid support for the assay. Theremoving step may be carried out by any suitable means, such as byincluding an enzyme in the solution which utilizes ATP as a substrate(e.g., including hexokinase and glucose in the aqueous solution in anamount sufficient so that essentially all of said ATP is utilized tophosphorylate the glucose; by including apyrase in the aqueous solutionin an amount effective to degrade the ATP; etc.). Alternatively, theremoving step may be carried out by washing the cell. The binding of thecompound to the P_(2U) receptor and activation of the receptor byagonists thereof may be detected by any suitable means, such asmeasuring for or detecting an elevation in intracellular calcium,measuring for or detecting an elevation in intracellular inositolphosphates, etc. Where only an indication of binding is desired,detection of binding may be carried out by any suitable means, such aswith a competitive binding assay with a known agonist or antagonist.

Vectors which are capable of expressing a P_(2U) receptor in a host cellare useful in methods of enhancing, facilitating, and/or acceleratingcell growth. Such methods involve the step of transforming a cell with avector capable of expressing a P_(2U) receptor in the cell in an amountsufficient to enhance cell growth. Cell growth is enhanced byautostimulation of the P_(2U) receptor, wherein the cell secretes ATPwhich activates the P_(2U) receptor, and wherein cell growth is enhancedby activation of the P_(2U) receptor (i.e., the binding of an agonistthereto). The vector may be as described above. The method is useful,among other things, for stimulating the growth of cultures of mammaliancells in vitro where the cells are difficult to grow or otherwise growslowly in culture.

Vectors which are capable of expressing a P_(2U) receptor in a host cellare also useful in methods of inhibiting or slowing cell growth. Suchmethods involve the step of transforming a cell with a vector capable ofexpressing a P_(2U) receptor in the cell in an amount sufficient toinhibit or slow cell growth. Cell growth is inhibited by autostimulationof the P_(2U) receptor, wherein the cell secretes ATP, and wherein cellgrowth is inhibited by activation of the P_(2U) receptor. The vector mayagain be as described above. The method is useful, among other things,for slowing the growth of cells in vitro where the cells are undulyproliferative, and for combatting proliferative or hyperplastic growthof cells or cell populations in a subject in need of such treatment,including both malignant and non-malignant cell growth (e.g., gobletcell hyperplasia).

Another aspect of the invention is antisense oligonucleotides (and DNAsencoding the same) having a sequence capable of binding specificallywith any sequences of an mRNA molecule which encodes a human P_(2U)receptor so as to prevent translation of the mRNA molecule (bindingconditions may be at the stringencies as given above with respect to DNAhybridization). This method may be carried out in any patient wheredecreasing mucus secretion by downregulating epithelial cell (e.g., lungairway epithelial cell) cell P_(2U) receptor expression is desired. Thismethod may also be carried out in vitro or in a subject in need of suchtreatment for the purpose of slowing cell growth for the purposesdescribed above. For example, in chronic bronchitis, the airway celldistribution is changed so that the number of goblet cells is increasedand the number of ciliated cells is decreased. This phenomenon, known asgoblet cell hyperplasia, is caused by chronic toxic exposure, and inpatients afflicted with this disorder a means for decreasing mucussecretion by inhibiting P_(2U) receptor expression in airway epithelialcells by administration of antisense nucleotides as described herein isdesired. Other examples where such treatment may be desired include thetreatment of gallblader epithelial mucos for cholecystitis and thetreatment of liver biliary ducts for cholangitis. Where the antisenseolgionucleotides are delivered directly to cells rather than via a DNAintermediate, nucleotides in which the phosphodiester bonds have beenmodified, e.g., to the methylphosphonate, the phosphotriester, thephosphorothioate, the phosphorodithioate, or the phosphoramidate, so asto render the oligonucleotide more stable in vivo may be used. Antisenseoligonucleotides may be of any suitable length (e.g., from about 8 or 10to 50 or 60 nucleotides in length), depending on the particular targetbeing bound and the mode of delivery thereof. The antisenseoligonucleotides are, in general, delivered or dosaged so thatintracellular levels of the antisense oligonucleotide of from 0.05 to 50μM are achieved.

Antisense oligonucleotides may be combined with an appropriatephysiologically or pharmaceutically acceptable carrier (e.g., sterilepyrogen-free physiological saline solution) to provide a pharmaceuticalformulation for delivery of the antisense oligonucleotide to cells, alsoin accordance with known techniques. See, e.g., U.S. Pat. No. 5,023,243to Tullis). The antisense oligonucleotide in the formulation may beenclosed within a liposome, microcrystal, or other lipid vesicle tofacilitate its being carried into the interior of cells, in accordancewith known techniques, as also discussed above. Antisenseoligonucleotides may also be delivered to cells by way of a DNAintermediate, as discussed in greater detail below.

In one embodiment, the present invention is carried out by administeringto the subject a vector carrying a nucleic acid active agent, whichvector is capable of entering epithelial mucosa cells (e.g., lung airwayepithelial mucosa cells). Such vectors may be formulated withpharmaceutical carriers and administered topically to the epithelialcells, such as by use of an aerosol delivery system in which respirableparticles comprising the active agent to be delivered to the cells aregenerated and delivered to the airway surfaces of the subject. Suitablevectors are typically viral vectors, including DNA viruses (wherein thenucleic acid active agent is DNA) and RNA viruses, or retroviruses(wherein the nucleic acid active agent is RNA). Techniques for carryingout gene therapy are known and for the delivery of genetic materials toepithelial mucosa such as lung airway epithelial mucosa are known. See,e.g., F. Collins et al., U.S. Pat. No. 5,240,846; I. Pastan, U.S. Pat.No. 5,166,059; R. Debs et al., PCT Application WO 93/12756; T.Friedmann, Science 244, 1275 (1989).

DNAs of the present invention, and oligonucleotides derived therefrom,are useful for screening for restriction fragment length polymorphism(RFLP) associated with disorders such as cystic fibrosis or otherdisorders potentially involving a defective P_(2U) receptor (ordefective regulation thereof).

The present invention is explained in greater detail in the followingExamples. These Examples are for illustrative purposes only, and are notto be taken as limiting of the invention.

EXAMPLE 1 Expression of Human P_(2U) Receptor in Human Astrocytoma Cells

I. EXPERIMENTAL PROCEDURES

cDNA Cloning and Sequencing. Two degenerate oligonucleotide primers,5'-AATGG(C/A/G)AC(C/T/A)TGGGA(G/A)GG(G/A)GA(C/T)GA(A/G)-3' (SEQ IDNO:5), and 5'-GACGTG(C/G)AA(A/G)GGCAG(A/C)(A/C)AGC(A/T)GAGGGCGAA-3' (SEQID NO:6), from the N-terminal and transmembrane VI domains,respectively, of the murine P2U receptor sequence (Lustig et al., Proc.Natl. Acad. Sci. USA 90, 5113 (1993)), were used in a polymerase chainreaction to amplify products from a cDNA library constructed in lambdaUni-ZAP XR (Stratagene, La Jolla, Calif.) from CF/T43 cell poly(A+) RNA.Amplification conditions were: 94° C., 1.5 min, 50° C., 1.5 min, 72° C.,2 min; 30 cycles. Products were cloned into pCR II (Invitrogen, SanDiego, Calif., USA) and screened by Southern blot using a randomprimer-labeled fragment corresponding to bases 290-1097 (amino acidresidues 8-276) of the murine P_(2U) sequence (probe P263).

A cloned PCR product of about 500 bases (probe D9) which hybridized withprobe P263 was labeled by random priming and used to screen 7×10⁵recombinants of the CF/T43 cDNA library. Hybridization was performedusing the QUIKHYB™ hybridization system (Stratagene) according to themanufacturer's instructions, except that 0.2×SSC/0.1% SDS was used forthe high stringency wash. One plaque was identified which hybridizedstrongly with probe D9. After purification of phage by additionalscreening, pbluescript SK(-) was rescued by in vivo excision(Stratagene). An HT-29 cDNA library prepared in lambda gt10 was screenedusing probe P263, and insert from positive plaque-purified phage wassubcloned into the Not I site of pbluescript. Plasmid DNA was purifiedby cesium chloride gradient centrifugation (Sambrook et al., MolecularCloning: A Laboratory Manual (2d ed. 1989)) and both strands sequencedby dideoxy termination (Sequenase version 2.0; (United StatesBiochemical Corp., Cleveland, Ohio, USA).

Probe P263 was generated by high stringency PCR amplification of themurine P_(2U) receptor clone, pP2R, obtained through the generosity ofK. Lustig and D. Julius (Univ. of California, San Francisco, Calif.,USA). The primers used to amplify P263 were: 5'-CTGGAATAGCACCATCAATGG-3'(SEQ ID NO:7) and 5'-GAGGTCAAGTGATCGGAAGG-3' (SEQ NO:8)

Heterologous Expression. A retroviral vector-containing plasmid,pLHP2USN, was constructed by insertion of the cloned CF/T43 cDNA intothe EcoRI and XhoI sites of pLXSN (A. Miller and G. Rosman,Biotechniques 7, 980 (1989)). An amphotrophic packaging cell line,PA317, was used to produce the LHP2USN retroviral vector and a controlvector containing Neor only (LSN). Clonal human astrocytoma cells1321N₁, UNC strain, generously provided by T. K. Harden (UNC, ChapelHill, N.C.), were plated at 150,000 cells per 60-mm dish and infectedwith LHP2USN or LSN (2 hr, 4 ug/ml Polybrene) and after 48 hr selectedwith 600 ug/ml G418 (Life Technologies, Inc., Gaithersburg, Md., USA).To assay for receptor activity, intracellular calcium concentration wasmeasured in confluent cells on coverslips by fura-2 fluorescence usingmicrospectrofluorimetry as previously described (S. Mason et al., Br. J.Pharmacol. 103, 1649 (1991)). Measurement of cell inositol phosphateformation was as described by H. Brown et al., Mol. Pharmacol. 40, 648(1991), in accordance with known techniques.

Northern and Southern Blots. Total cell and poly(A+) RNA were isolatedby standard procedures or purchased commercially (Clontech, Palo Alto,Calif., USA). Human and mouse genomic DNA was extracted from culturedcells (Sambrook et al., supra) and digested (5 U/μg DNA) with KpnI,EcoRI, Bam HI, or XbaI. RNA (formaldehyde denatured) or restrictionfragments of genomic DNA were resolved on agarose gels, transferred toNytran or nitrocellulose membranes (Schleicher & Schuell) as described(Sambrook et al., supra), and UV-crosslinked (Stratagene).Prehybridization and hybridization of blots with cDNA probes wereperformed using the QUIKHYB™ hybridization system (Stratagene) asdescribed above. Autoradiographs were done with Kodak XAR film and oneintensifying screen at -80° C.

II. RESULTS AND DISCUSSION

A cDNA fragment from a human airway (CF/T43) cDNA library, amplifiedusing degenerate primers based on the murine P2U receptor sequence, wasused to obtain a full-length plasmid clone, pHAP2U, from the samelibrary, which contains the insert sequence given herein as SEQ ID NO:1.Concomitantly, low stringency screening with the murine P_(2U) partialcDNA, P263, of an HT-29 cDNA library produced a clone which, uponsequence analysis, was found to contain cDNA identical to the pHAP2Uinsert. The deduced amino acid sequence of an open reading frame (SEQ IDNO: 1 and SEQ ID NO:2) present in the human cDNA bears substantialsimilarity (89% identity) to the murine P_(2U) receptor sequencereported by Lustig et al., supra, but is considerably less similar tothe chick P2Y purinoceptor (37% identity) described by T. Webb et al.,FEBS Lett. 324, 219 (1993).

The pHAP2U insert (HP2U) sequence exhibits structural features typicalof the family of G protein-coupled receptors and common to both thechick and murine P₂ -receptors. These include: (1) seven hydrophobicdomains, (2) consensus N-linked glycosylation sequences near theN-terminus, (3) a number of residues highly conserved among Gprotein-coupled receptors (e.g., Asn51, Asp79, Cys106 and Cys183), and(4) potential phosphorylation sites in the third intracellular andC-terminal domains. The putative P_(2U) receptor sequences also have apotential site for phosphorylation by protein kinase C in the secondintracellular loop. Alignment of the human and murine P_(2U) receptorsequences reveals 4 additional residues, as well as a number ofmismatched residues, in the C-terminal intracellular domain of the humanclone. Like the murine and chick P₂ -receptors, the human P_(2U)receptor sequence does not appear to be closely related to any of theother cloned G protein-coupled receptors, including those for adenosineand cAMP.

A retrovirus expression system was employed to study the cloned CF/T43HP2U cDNA in 1321N1 human astrocytoma cells. The effect of extracellularnuceotides on intracellular calcium, as measured by fura-2 fluorescence,was assessed in CF/T43 cells, uninfected 1321N1 cells, and 1321N1 cellsinfected with retroviral vector containing either the cloned HP2U or noinsert (LSN). Exposure of CF/T43 cells to extracellular UTP (10⁻⁴ M)resulted in an initial sharp increase in Ca²⁺ !_(i) which relaxed over1-2 min to a prolonged plateau (data not shown). Application of UTP toCF/T43 cells in Ca₂₊ -free medium induced a sharp increase in Ca²⁺ !_(i)which returned to baseline without an intervening plateau phase. Incontrast, extracellular UTP had no effect on uninfected 1321N1 cells(data not shown) or LSN-infected cells (data not shown), whereascarbachol elicited responses in each case. However, the Ca²⁺ !_(i)response of 1321N1 cells expressing the LHP2USN vector to UTP wassimilar to that of CF/T43 cells in both Ca₂₊ -free and Ca₂₊ -containingmedia (data not shown). In each experiment, the addition of ATP producedthe same result observed with UTP. A response to extracellularnuceotides was seen in all trials with CF/T43 (peak Ca²⁺ !_(i) =750+160nM, n=10) and LHP2USN-expressing 1321N1 cells (peak Ca²⁺ !_(i) =850+170nM, n=6) but not with uninfected or LSN-infected controls.

To examine the pharmacological specifity of the HP2U clone expressed in1321N1 cells, concentration-effect curves were generated for nucleotidesand structural analogues active at P_(2X), P_(2Y) and P_(2U) receptors(data not shown). UTP and ATP were equipotent, with EC₅₀ values of 1.4and 1.0 uM, respectively, but UTP was slightly more efficacious. TheP_(2Y) agonist, 2MeSATP, and P_(2X) agonist, α,β-MeATP, had littleeffect. These concentration-effect relations are very similar to thosereported for heterologously expressed murine P_(2U) (Lustig, et al.,supra) and virtually indistinguishable from data (not shown) generatedfor the endogenous CF/T43 and HT-29 receptors. Clearly, the agonistspecifities fit the defininition of the P_(2U) receptor.

Pretreatment of CF/T43 and HP2U-1321N1 cells with pertussis toxininhibited ATP-stimulated intracellular Ca²⁺ mobilization in bothcell-types by 20-30% (data not shown). These results are consistent withthe effects of pertussis toxin on inositol phosphate accumulationinduced by ATP or UTP in CF/T43 cells (Brown et al., supra (1991)), andon P_(2U) responses in other systems (Murphy and Tiffany, J. Biol. Chem.265, 11615 (1990)).

The pertussis toxin sensitivity of HP2U-1321N1 cells, along with theobservation that UTP/ATP raised Ca²⁺ !_(i) in the absence ofextracellular Ca²⁺, suggest that HP_(2U) is coupled to phospholipase C(PLC) via a G protein. To test this possibility more directly, wemeasured inositol phosphate formation. When these studies were firstperformed, very high levels of inositol phosphates were found toaccumulate in HP_(2U) -1321N1 cells which had not been exposed toexogenous nucleotides (FIG. 1A). We hypothesized that during the lengthylabeling period 1321N1 cells released 5'-nucleotides into the medium inquantities sufficient to "self-activate" the receptor. The inclusion ofapyrase in the medium during the cell-labeling step reduced the baselineinositol phosphate levels to values near those of controls (FIG. 1B).Thus, it appears the cells are persistently stimulated by nucleotides inan autocrine fashion. The mode of release of nucleotides from theastrocytoma cells and the biological consequences of the persistentautocrine activation are unknown. To facilitate comparison withcontrols, the inositol phosphate studies were performed on cellspretreated with apyrase. Consistent with coupling of the human P_(2U)receptor to PLC, ATP and UTP increased inositol phosphate accumulationin 1321N1 cells expressing LHP_(2U) SN but not in uninfected orLSN-infected controls (data not shown).

P_(2U) receptor mRNA is widely distributed in human tissue: transcriptswere detected in heart, liver, lung, and kidney, as reported for themouse (Lustig et al., supra 1993), as well as placenta and skeletalmuscle (data not shown). In addition, it was present in primary culturesof human respiratory epithelia and kidney proximal tubules, and inHSG-Pa salivary gland duct, CFT1 airway, and T84 colon cell lines.

Several of the human tissues and cell lines were found to express morethan one mRNA. Transcripts of 2.1 kb, 7.5 kb, and 9 kb were observed,and in some tissues all three RNAs were present. Although all of thebands in human tissues were found to cross hybridize with a full lengthmurine P_(2U) receptor cDNA, none of the murine tissues analyzed byLustig et al., supra, or mouse skeletal muscle contain more than onetranscript. Human liver and other human cells, including primarycultures of proximal tubule epithelium, were found to express only the2.1 kb message, but both CF/T43 and HT-29 cells express at least twotranscripts.

Although Murphy and Tiffany, supra, have reported that HL60 cells mayexpress more than one mRNA coding for functional P_(2U) receptors, thesignificance of multiple transcripts is unknown. If all of the bandsrepresent fully processed mRNAs they may be alternatively processedforms of the same gene or products of different genes. Alternativeprocessing might produce transcripts that differ in their noncodingregions but code for identical proteins. On the other hand, since thestructure of P₂ -receptor genes is unknown, the possibility remains thatthe different RNAs code for functionally different proteins, especiallyin light of the evidence presented below which suggests that at leasttwo genes for P_(2U) receptors exist in the human genome.

Human genomic DNA was digested with restriction enzymes known not to cutin the cDNA fragments used as probes. Under stringent conditions, ahuman P_(2U) receptor coding sequence probe, D9, recognized two discretebands in human genomic DNA cut with Kpn I, Eco RI, and Xba I, and threebands in DNA cut with Bam HI. The blot was then stripped and reprobedwith a Kpn I fragment containing a segment of the 3' most bases of theHP_(2U) clone. If the bands are portions of the same gene, only onewould be associated with the 3' portion of the gene. In contrast, theHP_(2U) Kpn I fragment recognized two bands in all of the digests (datanot shown) suggesting that these bands are separate genes and notportions of the same gene. The downward shift of the more intenselyhybridizing band in the Kpn I digest is consistent with the presence ofa Kpn I site between the sequences recognized by the two probes. Thelack of a shift in the position of the less intensely hybridizing bandof the Kpn I digest and the loss of one of three bands in the Bam HIdigest suggest that the second gene has lost the Kpn I site and that aBam HI site has arisen in the portion recognized by the coding sequenceprobe. Both findings support the hypothesis that the probes arehybridizing to two unique genes.

A fragment from the coding region of the murine P_(2U) receptor cDNA,probe P263, recognized a single band in mouse genomic DNA digested witheither Eco RI or Bam HI. Whether these results correlate with theobservation that human tissues appear to express more than one speciesof P_(2U) message, while only one P_(2U) receptor mRNA was found inmouse tissues, remains to be resolved.

EXAMPLE 2 Expression of Murine P_(2U) Receptor in Human Leukemia Cells

I. EXPERIMENTAL PROCEDURES

Materials. pRc/CMV was purchased from Invitrogen (San Diego, Calif.).K562 human leukemia cells (CCL-243) were obtained from American TypeCulture Collection (Rockville, Md.). Geneticin (G418) was purchased fromGIBCO (Grand Island, N.Y.). Electroporation cuvettes (0.4 cm electrodegap) and the GENE PULSER™ electroporation chamber were obtained fromBiorad (Richmond, Calif.). NYTRAN™ nylon membranes were purchased fromSchleicher and Schuell (Keene, N.J.). α-³² P!ATP and α-³² P!UTP wereobtained from ICN Radiochemicals (Irvine, Calif.). AVICEL™microcrystalline silica gel thin layer chromatography plates containinga fluorescent indicator for nucleotides were obtained from Analtech(Newark, Del.). N,N-dimethylformamide and 4-benzoylbenzoic acid wereobtained from Aldrich (Milwaukee, Wis.). HIS-BIND™ resin was purchasedfrom Novagen (Madison, Wis.). ATPγS was purchased from BoehringerMannheim (Indianapolis, Ind.) and 2-methylthioATP was purchased fromResearch Biochemicals Inc. (Natick, Mass.). Other reagents werepurchased from Sigma (St. Louis, Mo.). Concentrations of nucleotidestock solutions were verified spectrophotometrically.

Cell Culture. K562 cells were cultured in spinner flasks (58 rpm) ingrowth medium (pH 7.4) composed of RPMI-1640, 10% heat-inactivated fetalbovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 μg/mlstreptomycin. The cell suspensions were maintained at 37° C. in ahumidified atmosphere of 5% CO₂ and 95% air at a density of 1×10⁵ to1×10⁶ cells/ml.

Stable and Transient Transfections. To generate stable transfectantsexpressing the P_(2U) receptor, K562 cells were transfected byelectroporation (F. Toneguzzo et al., Mol. Cell. Biol. 6, 703 (1986))with the neomycin-resistance plasmid pP2R1. pP2R1 is pRc/CMV containinga ˜2.4 kb HindIII-NotI fragment of the P_(2U) receptor cDNA, missing thefirst 6 nucleotides of the 5' untranslated region of the full lengthcDNA. control cells were stably transfected with pRc/CMV. Cells weresedimented at 200×g for 5 min at room temperature, rinsed once withphosphate-buffered saline (10 mM NaH₂ PO₄, pH 7.4, 120 mM NaCl, 5 mMKCl), and resuspended in phosphate-buffered saline at a finalconcentration of 4×10⁷ cells/ml. The cells were chilled on ice for 10min and then transferred in 0.5 ml aliquots to prechilledelectroporation cuvettes. DNA (40 μg of pP2R1 linearized with PvuI) wasadded to the cell suspension, the cells were subjected to a singleelectrical pulse (1 kV, 25 μF) and the cuvettes were placed on ice.After 10 min, growth medium was added and the cells were incubated at37° C. Fresh growth medium supplemented with 0.7 mg/ml G418 was added 48h later, and the cells were incubated for 2 weeks to select for stabletransfectants expressing neomycin resistance. Clonal cell lines werethen isolated by transferring single cells to individual wells of a96-well culture plate: each well contained 0.2 ml of conditioned growthmedium filtered from K562 stock cultures, 0.2 ml of fresh growth medium,and 0.4 mg/ml G418. The clonal cell lines were cultured for three tofour weeks in growth medium containing 0.4 mg/ml G418 and assayed forP_(2U) receptor, the results shown are from one representative clonalcell line.

To generate transient transfectants, K562 cells were transfected asabove with 40 μg unlinearized plasmid pCCM6×1 or pP2R (Lustig et al.,supra 1993). Cells were incubated for 48 h at 37° C. in 100 mm culturedishes containing 12 ml of growth medium and assayed for P_(2U) receptoractivity.

Cytoplasmic Free Calcium Measurements. Cells were sedimented at 200×gfor 5 min at room temperature and washed once with 10 ml ofHepes-buffered saline (20 mM Hepes, pH 7.4, 120 mM NaCl, 5 mM KCl, 1 mMMgCl₂, and 1 mM CaCl₂). Washed cells were suspended in Hepes-bufferedsaline at 5×10⁵ cells/ml, and stored (for up to 4 h) at 37° C. Theconcentration of cytoplasmic free calcium, Ca²⁺ !_(i), was measured bydual-excitation spectrofluorometric analysis of cells loaded withfura-2, a fluorescent probe for calcium.

Northern analysis. Poly (A)⁺ RNA was isolated from K562 cells with aFastTrack® mRNA isolation kit (Invitrogen) and electrophoresed in 0.8%agarose-6.5% formaldehyde gels. The mRNA was transferred by capillaryflow to a nylon membrane, prehybridized for 15 min at 68° C. inQuick-Hyb®(Stratagene), and hybridized for 1 h at 68° C. withrandom-primer labeled DNA synthesized in vitro from the Hind III-Not Ifragment of pP2R. The membrane was washed twice at room temperature in 2X SSC containing 0.1% SDS, once at 60° C. in 0.2 X SSC containing 0.1%SDS, and exposed to Kodak XAR film with an intensifying screen for 24 hrat -70° C.

Synthesis of α-³² P!BzATP and α-³² P!BZUTP. The following procedure wascarried out in dim rom light to avoid photoactivation of thebenzophenone moiety. 18 mg of 4-benzoylbenzoic acid (BzBz) and 42 mg of1,1'-carbonyldiimidazole were dissolved in 100 μl ofN,N-dimethylformamide and stirred in a tightly stoppered 5 mlpolypropylene test tube for 15 min at room temperature. Then, 0.5 ml of3.3 μM α-³² P!ATP (3000 Ci/mmol) was added slowly and the reactionmixture was stirred overnight (approx. 15 h) at room temperature in aloosely stopered test tube. The reaction mixture was centrifuged at2000×g for 5 min and the supernatant was added to a Sephadex LH-20column (1.5×45 cm) and eluted with 100 mM ammonium formate (pH 7.4) at aflow rate of 0.7 ml/min. The eluent was collected in 3.0 ml fractionsand the fractions containing radioactivity were detected with a Geigercounter. Fractions 30-48 comprising the second radioactive peak ( α-³²P!BzATP) were pooled, lyophilized, reconstituted in 1 ml of H₂ O andstored at -70° C. The amount of radioactivity in the α-³² P!BzATP samplewas determined by liquid scintillation spectrometry and theconcentration of α-³² P!BzATP and α-³² P!ATP were equal. A 10-15% BzATPyield was obtained relative to the starting concentration of ATP. Thepurity of the α-³² P!BzATP sample was assessed by thin layerchromatography (TLC) on silica gel plates containing a fluorescentindicator for nucleotides. A TLC plate was spotted with 1 μl of the α-³²P!BzATP sample and other lanes were spotted with 5 nanomoles ofunlabeled ATP, BzATP (Sigma Chemical Co.) and BzBz. The plate wasdeveloped for 2 h at room temperature in a chromatography chambercontaining 100 ml of 1-butanol:H₂ O:glacial acetic acid (5:3:2, v/v/v)and the nonradioactive standards were visualized by exposing the plateto short wavelength ultraviolet light. The R_(f) values for ATP, BzATPand BzBz were 0.17, 0.65 and 0.98, respectively. An autoradiograph ofthe TLC plate indicated that the α-³² P!BzATP sample was >95% free ofα-³² P!ATP contamination. Synthesis of α-³² P!BzUTP was performed as forα-³² P!BzATP except that 0.5 ml of 3.3 μM α-³² P!UTP (3000 Ci/mmol) wassubstituted for α-³² P!ATP in the reaction mixture. The R_(f) values ofUTP and BzUTP were 0.21 and 0.70, respectively.

Construction and Purification of the Histidine-tagged P_(2U) Receptor(P2HIS). To produce P2HIS, 70 pmoles of the primers CGCACCATTGCCTTGGTAC(SEQ ID NO:3) (which contains a BstXI site) andGAGGATATCCTAATGATGATGATGATGATGTAGCCGAATGTCCTTAGTCTC (SEQ ID NO:4) (whichcontains an EcoRV site, stop codon, and the hexahistidine codingsequence) were incubated with 50 ng of pP2R, 20 nmoles of each dNTP, 1Xreaction buffer (20 mM Tris-HCl pH 8.2, 10 mM KCl, 6 mM (NH₄)₂ SO₄, 1.5mM MgCl₂, and 0.1% Triton X-100), and H₂ O to bring the reaction mixtureto a 100 μl volume. The reaction mixture was placed in a thermo-cyclerand heated to 95° C. for 5 min prior to the addition of 2.5 units of PƒuDNA polymerase (Stratagene), a thermostable enzyme that possesses both5' to 3' polymerase and 3' to 5' proofreading exonuclease activities.The reaction mixture was overlayed with 50 μl of mineral oil andsubjected to 30 PCR cycles (95° C. for 1.5 min, 55° C. for 1.5 min, and72° C. for 2 min). The PCR product, a single 400 bp band, was subclonedinto the unique BstXI-EcoRV restriction site of pP2R to generate pP2HIS.K562 cells with stable P2HIS expression were produced by subcloning theHindIII-NotI restriction fragment of pP2HIS encoding the histidine-tagedP_(2U) receptor cDNA into pRC/CMV and transfecting K562 cells asdescribed for pP2R1. The composition of pP2HIS was verified by DNAsequence analysis.

To isolate P2HIS, plasma membranes from 10⁸ K562 cells with stable P2HISexpression were purified according to procedure of Thom et al. Biochem.J. 168, 187 (1977). The P_(2U) receptors were then photolabeled byincubating 1 mg of plasma membrane protein in 2 ml of Hepes-EDTA buffer(20 mM Hepes, pH 7.5, 1mM EDTA, 0.1 mM phenylmethylsulfonylfluoride, and0.1 mM benzamidine) containing 5 nM α-³² P!BzATP or 5 nM α-³² P!BzUTPfor 5 min at 37° C. followed by irradiation with long wavelength UVlight for 15 min at 4° C. The irradiated membranes were washed twicewith Hepes-EDTA buffer, pelleted by centrifugation at 35,000×g for 10min at 4° C., and solubilized in 1 ml of urea buffer (6M urea, 500 mMNaCl, 20 mM Tris-HCl, pH 7.9). The solubilized proteins were applied toa Ni²⁺ -charged Sepharose column (2.5 ml bed volume) and eluted with animidazole gradient. Column eluents were dialyzed against 10 mM Tris-HCl(pH 7.4), lyophilized, and resolubilized in 50 μl of SDS-sample buffer.The samples were electrophoresed on a 5-16% SDS-polyacrylamide gel forapproximately 16 h at 5 milliamps. The gel was dried and exposed toautoradiographic film for 1 to 3 days in order to visualize theradiolabeled proteins.

II. RESULTS AND DISCUSSION

ATP or UTP produced a two-fold increase in Ca²⁺ !_(i) in K562 cellsstably or transiently transfected with P_(2U) receptor cDNA (data notshown). Ca²⁺ !_(i) was maximal within 5 s after nucleotide addition andthen returned to the basal level within 1 min. Neither ATP nor UTPelevated Ca₂₊ !^(i) in untransfected K562 cells or in vector-transfectedK562 cells. In transient transfectants, the largest P_(2U)receptor-mediated increases in Ca²⁺ !^(i) were observed 48 h after thetransfection procedure.

In stable transfectants, the ATP- and UTP-mediated increases in Ca²⁺!^(i) were not affected by depletion of extracellular Ca²⁺ by EGTA (datanot shown), suggesting that P_(2U) receptor activation causes themobilization of intracellular calcium and not the influx ofextracellular calcium. Consistent with this idea, ATP and UTP alsotransiently reduced intracellular levels of phosphatidylinositol4,5-bisphosphate (PIP₂) in the stable transfectants by approximately10%. Activation of endogenous P_(2U) receptors in NG108-15 cellssimilarly leads to the phospholipase C-dependent hydrolysis of PIP₂, thegeneration of inositol 1,4,5-triphosphate (IP₃), and the mobilization ofcalcium from intracellular stores (Lin et al., J. Neurochem. 60, 1115(1993)). The relatively weak effect of ATP and UTP on PIP₂ hydrolysisand calcium mobilization in K562 cells transfected with P_(2U) receptorcDNA as compared to NG108-15 cells may reflect the low levels of IP₃receptors in undifferentiated K562 cells.

It has been controversial whether ATP and UTP interact with the samereceptor or whether the effect of each nucleotide is mediated byseparate receptors. In stable transfectants, the EC₅₀ values for ATP-and UTP-mediated increases in Ca²⁺ !_(i) were about 1.4 μM and about 0.8μM, respectively (FIG. 2). These responses are similar to those obtainedin Xenopus oocytes expressing the P_(2U) receptor where EC₅₀ values ofabout 1 μM were obtained for both nucleotides (Lustig et al., supra1993). These findings demonstrate conclusively that the product of asingle gene confers upon cells the ability to respond to both ATP andUTP.

K562 cells do not express an endogenous P_(2U) receptor but do express acalcium-mobilizing P_(2T) receptor that is activated by ADP andinhibited by ATP. In untransfected K562 cells, activation of the P_(2T)receptor by ADP or 2-methylthioATP, but not 1 mMATP, UTP, ATPγS, BzATPor BzUTP, elevated Ca²⁺ !_(i) (data not shown). ADP and 2-MeSATP do notappear to act as agonists of the P_(2U) receptor, since the doseresponse curves for these nucleotides were virtually identical in stabletransfectants and in vector-transfected K562 cells (FIG. 2). Similarly,ADP and 2-methylthioATP did not stimulate ⁴⁵ Ca²⁺ efflux in oocytesexpressing the cloned receptor (Lustig et al., supra 1993). Hydrolysisof ATP may not be required for P_(2U) receptor activation since theslowly-hydrolyzable ATP analog ATPγS was a relatively potent agonist(EC₅₀ =about 9.6 μM) of the P_(2U) receptor in stable transfectants(FIG. 2) and in Xenopus oocytes (Lustig et al., supra 1993).Furthermore, β, γ methyleneATP, β,γ imidoATP, GTP, TTP, CTP, UDP, AMPand adenosine, at concentrations as high as 1 mM, had no effect on Ca²⁺!_(i) ; in untransfected K562 cells or stable transfectants, and did notprevent ATP-mediated increases in Ca²⁺ !_(i) in stable transfectants(not shown). Thus, these compounds do not appear to act as agonists orantagonists of the P_(2U) receptor.

The photoaffinity probes 3'-O-(4-benzoyl) benzoyl adenosine5'-triphosphate (BzATP) and 3'-O -(4-benzoyl) benzoyl uridine5'-triphosphate (BzUTP) also were agonists of the P_(2U) receptor (FIG.2), although relatively weak agonists compared to ATP and UTP. BzATP haspreviously been used as an agonist and/or a photoaffinity probe forP_(2Z) receptors in transformed mouse fibroblasts, and mousemacrophages, P_(2Y) receptors in turkey erythrocytes, and P_(2U)receptors in bovine endothelial cells and NG108-15 cells. This, however,is the first report of BzUTP as a P₂ receptor agonist.

Isolation of the P_(2U) receptor and conclusive identification of itsmolecular weight were accomplished with the aid of a hexahistidine tagattached to the carboxy terminus of the P_(2U) receptor protein (P2HIS).Both ATP and UTP caused an increase in Ca²⁺ !_(i) and a decrease in PIP₂(not shown) in K562 cells that stably express P2HIS, indicating that theagonist specificity and signaling properties of the P_(2U) receptor wereunaltered by the hexahistidine tag. P2HIS was isolated by affinitychromatography on Ni²⁺ -charged Sepharose columns. To increase detectionof P2HIS, plasma membrane proteins from stable P2HIS transfectants werephotolabeled with α-³² P!BzATP or α-³² P!BzUTP. The results indicatedthat a 53 kDa protein was selectively retained on the column until veryhigh imidazole concentrations (200 mM) were used. The calculatedmolecular mass of the NG108-15 P_(2U) receptor is 42 kDa, suggestingthat the mature receptor is glycosylated on one or both of two N-linkedglycosyation consensus sites located in its putative amino-terminalextracellular domain.

The P_(2U) receptor cDNA encodes an mRNA of ˜2.4kb in a variety oftissues and cell lines (Lustig et al, supra 1993). Northern blotanalysis revealed that a P_(2U) receptor cDNA probe hybridized with an˜2.5 kb mRNA species in K562 cells that were transfected with P2R1 butdid not hybridize with any transcript in untransfected K562 cells (datanot shown). This suggests that the endogenous P_(2T) receptor in K562cells is not significantly homologous to the P_(2U) receptor.

These results indicate that we have expressed a functional 53 kDa P_(2U)receptor in K562 human leukemia cells. In stable and transienttransfectants, the P_(2U) receptor had the same agonist selectivity andsignaling properties as in Xenopus oocytes and in NG108-15 cells, thesource of the P_(2U) receptor cDNA. Thus, our findings define amammalian model system for rapidly analyzing structure-functionrelationships, radioligand-binding and signal transduction properties ofboth wild-type and mutant P_(2U) receptors.

The foregoing examples are illustrative of the present invention, andare not to be construed as limiting thereof. The invention is defined bythe following claims, with equivalents of the claims to be includedtherein.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 8                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1842 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 57..1181                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGAACCCGTGCAGGCGCTGAGCATCCTGACCTGGAGAGCAGGGGCTGGTCAGGGCG56                    ATGGCAGCAGACCTGGGCCCCTGGAATGACACCATCAATGGCACCTGG104                           MetAlaAlaAspLeuGlyProTrpAsnAspThrIleAsnGlyThrTrp                              151015                                                                        GATGGGGATGAGCTGGGCTACAGGTGCCGCTTCAACGAGGACTTCAAG152                           AspGlyAspGluLeuGlyTyrArgCysArgPheAsnGluAspPheLys                              202530                                                                        TACGTGCTGCTGCCTGTGTCCTACGGCGTGGTGTGCGTGCTTGGGCTG200                           TyrValLeuLeuProValSerTyrGlyValValCysValLeuGlyLeu                              354045                                                                        TGTCTGAACGCCGTGGGCCTCTACATCTTCTTGTGCCGCCTCAAGACC248                           CysLeuAsnAlaValGlyLeuTyrIlePheLeuCysArgLeuLysThr                              505560                                                                        TGGAATGCGTCCACCACATATATGTTCCACCTGGCTGTGTCTGATGCA296                           TrpAsnAlaSerThrThrTyrMetPheHisLeuAlaValSerAspAla                              65707580                                                                      CTGTATGCGGCCTCCCTGCCGCTGCTGGTCTATTACTACGCCCGCGGC344                           LeuTyrAlaAlaSerLeuProLeuLeuValTyrTyrTyrAlaArgGly                              859095                                                                        GACCACTGGCCCTTCAGCACGGTGCTCTGCAAGCTGGTGCGCTTCCTC392                           AspHisTrpProPheSerThrValLeuCysLysLeuValArgPheLeu                              100105110                                                                     TTCTACACCAACCTTTACTGCAGCATCCTCTTCCTCACCTGCATCAGC440                           PheTyrThrAsnLeuTyrCysSerIleLeuPheLeuThrCysIleSer                              115120125                                                                     GTGCACCGGTGTCTGGGCGTCTTACGACCTCTGCGCTCCCTGCGCTGG488                           ValHisArgCysLeuGlyValLeuArgProLeuArgSerLeuArgTrp                              130135140                                                                     GGCCGGGCCCGCTACGCTCGCCGGGTGGCCGGGGCCGTGTGGGTGTTG536                           GlyArgAlaArgTyrAlaArgArgValAlaGlyAlaValTrpValLeu                              145150155160                                                                  GTGCTGGCCTGCCAGGCCCCCGTGCTCTACTTTGTCACCACCAGCGCG584                           ValLeuAlaCysGlnAlaProValLeuTyrPheValThrThrSerAla                              165170175                                                                     CGCGGGCCGCTAACCTGCCACGACACCTCGGCACCCGAGCTCTTCAGC632                           ArgGlyProLeuThrCysHisAspThrSerAlaProGluLeuPheSer                              180185190                                                                     CGCTTCGTGGCCTACAGCTCAGTCATGCTGGGCCTGCTCTTCGCGGTG680                           ArgPheValAlaTyrSerSerValMetLeuGlyLeuLeuPheAlaVal                              195200205                                                                     CCCTTTGCCGTCATCCTTGTCTGTTACGTGCTCATGGCTCGGCGACTG728                           ProPheAlaValIleLeuValCysTyrValLeuMetAlaArgArgLeu                              210215220                                                                     CTAAAGCCAGCCTACGGGACCTCGGGCGGCCTGCCTAGGGCCAAGCGC776                           LeuLysProAlaTyrGlyThrSerGlyGlyLeuProArgAlaLysArg                              225230235240                                                                  AAGTCCGTGCGCACCATCGCCGTGGTGCTGGCTGTCTTCGCCCTCTGC824                           LysSerValArgThrIleAlaValValLeuAlaValPheAlaLeuCys                              245250255                                                                     TTCCTGCCATTCCACGTCACCCGCACCCTCTACTACTCCTTCCGCTCG872                           PheLeuProPheHisValThrArgThrLeuTyrTyrSerPheArgSer                              260265270                                                                     CTGGACCTCAGCTGCCACACCCTCAACGCCATCAACATGGCCTACAAG920                           LeuAspLeuSerCysHisThrLeuAsnAlaIleAsnMetAlaTyrLys                              275280285                                                                     GTTACCCGGCTGGCCAGTGCTAACAGTTGCCTTGACCCCGTGCTCTAC968                           ValThrArgLeuAlaSerAlaAsnSerCysLeuAspProValLeuTyr                              290295300                                                                     TTCCTGGCTGGGCAGAGGCTCGTACGCTTTGCCCGAGATGCCAAGCCA1016                          PheLeuAlaGlyGlnArgLeuValArgPheAlaArgAspAlaLysPro                              305310315320                                                                  CCCACTGGCCCCAGCCCTGCCACCCCGGCTCGCCGCACGCTGGGCCTG1064                          ProThrGlyProSerProAlaThrProAlaArgArgThrLeuGlyLeu                              325330335                                                                     CGCAGATCCGACAGAACTGACATGCAGAGGATAGGAGATGTGTTGGGC1112                          ArgArgSerAspArgThrAspMetGlnArgIleGlyAspValLeuGly                              340345350                                                                     AGCAGTGAGGACTCTAGGCGGACAGAGTCCACGCCGGCTGGTAGCGAG1160                          SerSerGluAspSerArgArgThrGluSerThrProAlaGlySerGlu                              355360365                                                                     AACACTAAGGACATTCGGCTGTAGGAGCAGAACACTTCAGCCTGTGCAGGT1211                       AsnThrLysAspIleArgLeu                                                         370375                                                                        TTATATTGGGAAGCTGTAGAGGACCAGGACTTGTGCAGACGCCACAGTCTCCCCAGATAT1271              GGACCATCAGTGACTCATGCTGGATGACCCCATGCTCCGTCATTTGACAGGGGCTCAGGA1331              TATTCACTCTGTGGTCCAGAGTCAACTGTTCCCATAACCCCTAGTCATCGTTTGTGTGTA1391              TAAGTTGGGGGAATTAAGTTTCAAGAAAGGCAAGAGCTCAAGGTCAATGACACCCCTGGC1451              CTGACTCCCATGCAAGTAGCTGGCTGTACTGCCAAGGTACCTAGGTTGGAGTCCAGCCTA1511              ATCAAGTCAAATGGAGAAACAGGCCCAGAGAGGAAGGTGGCTTACCAAGATCACATACCA1571              GAGTCTGGAGCTGAGCTACCTGGGGTGGGGGCCAAGTCACAGGTTGGCCAGAAAACCCTG1631              GTAAGTAATGAGGGCTGAGTTTGCACAGTGGTCTGGAATGGACTGGGTGCCACGGTGGAC1691              TTAGCTCTGAGGAGTACCCCCAGCCCAAGAGATGAACATCTGGGGACTAATATCAATAGA1751              CCCATCTGGAGGCTCCCATGGGCTAGGAGCCAGTGTGAGGCTGTAACTTATACTAAAGGT1811              TGTGTTGCCTGCTAAAAAAAAAAAAAAAAAA1842                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 375 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAlaAlaAspLeuGlyProTrpAsnAspThrIleAsnGlyThrTrp                              151015                                                                        AspGlyAspGluLeuGlyTyrArgCysArgPheAsnGluAspPheLys                              202530                                                                        TyrValLeuLeuProValSerTyrGlyValValCysValLeuGlyLeu                              354045                                                                        CysLeuAsnAlaValGlyLeuTyrIlePheLeuCysArgLeuLysThr                              505560                                                                        TrpAsnAlaSerThrThrTyrMetPheHisLeuAlaValSerAspAla                              65707580                                                                      LeuTyrAlaAlaSerLeuProLeuLeuValTyrTyrTyrAlaArgGly                              859095                                                                        AspHisTrpProPheSerThrValLeuCysLysLeuValArgPheLeu                              100105110                                                                     PheTyrThrAsnLeuTyrCysSerIleLeuPheLeuThrCysIleSer                              115120125                                                                     ValHisArgCysLeuGlyValLeuArgProLeuArgSerLeuArgTrp                              130135140                                                                     GlyArgAlaArgTyrAlaArgArgValAlaGlyAlaValTrpValLeu                              145150155160                                                                  ValLeuAlaCysGlnAlaProValLeuTyrPheValThrThrSerAla                              165170175                                                                     ArgGlyProLeuThrCysHisAspThrSerAlaProGluLeuPheSer                              180185190                                                                     ArgPheValAlaTyrSerSerValMetLeuGlyLeuLeuPheAlaVal                              195200205                                                                     ProPheAlaValIleLeuValCysTyrValLeuMetAlaArgArgLeu                              210215220                                                                     LeuLysProAlaTyrGlyThrSerGlyGlyLeuProArgAlaLysArg                              225230235240                                                                  LysSerValArgThrIleAlaValValLeuAlaValPheAlaLeuCys                              245250255                                                                     PheLeuProPheHisValThrArgThrLeuTyrTyrSerPheArgSer                              260265270                                                                     LeuAspLeuSerCysHisThrLeuAsnAlaIleAsnMetAlaTyrLys                              275280285                                                                     ValThrArgLeuAlaSerAlaAsnSerCysLeuAspProValLeuTyr                              290295300                                                                     PheLeuAlaGlyGlnArgLeuValArgPheAlaArgAspAlaLysPro                              305310315320                                                                  ProThrGlyProSerProAlaThrProAlaArgArgThrLeuGlyLeu                              325330335                                                                     ArgArgSerAspArgThrAspMetGlnArgIleGlyAspValLeuGly                              340345350                                                                     SerSerGluAspSerArgArgThrGluSerThrProAlaGlySerGlu                              355360365                                                                     AsnThrLysAspIleArgLeu                                                         370375                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CGCACCATTGCCTTGGTAC19                                                         (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GAGGATATCCTAATGATGATGATGATGATGTAGCCGAATGTCCTTAGTCTC51                         (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AATGGVACHTGGGARGGRGAYGAR24                                                    (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GACGTGSAARGGCAGMMAGCWGAGGGCGAA30                                              (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CTGGAATAGCACCATCAATGG21                                                       (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GAGGTCAAGTGATCGGAAGG20                                                        __________________________________________________________________________

That which is claimed is:
 1. A method of inhibiting the growth, of acell in culture comprising:transforming a mammalian cell in culture witha vector that expresses a P_(2U) receptor in said cell in an amountsufficient to inhibit cell growth by autostimulation of said P_(2U)receptor, wherein said vector contains and expresses DNA encoding ahuman P_(2U) receptor selected from the group consisting of;(a) isolatedDNA having SEQ ID NO:1; and (b) isolated DNA differing from the isolatedDNA of (a) above in nucleotide sequence due to the degeneracy of thegenetic code; wherein said cell secretes ATP or UTP, and wherein cellgrowth is inhibited by stimulation of said P_(2U) receptor with ATP orUTP.